Antibody recognition of rodent malaria parasite antigens exposed at the infected erythrocyte surface: specificity of immunity generated in hyperimmune mice

In regions where malaria is endemic, inhabitants remain susceptible to repeated reinfection as they develop and maintain clinical immunity. This immunity includes responses to surface-exposed antigens on Plasmodium sp.-infected erythrocytes. Some of these parasite-encoded antigens may be diverse and phenotypically variable, and the ability to respond to this diversity and variability is an important component of acquired immunity. Characterizing the relative specificities of antibody responses during the acquisition of immunity and in hyperimmune individuals is thus an important adjunct to vaccine research. This is logistically difficult to do in the field but is relatively easily carried out in animal models. Infections in inbred mice with rodent malaria parasite Plasmodium chabaudi chabaudi AS represent a good model for Plasmodium falciparum in humans. This model has been used in the present study in a comparative analysis of cross-reactive and specific immune responses in rodent malaria. CBA/Ca mice were rendered hyperimmune to P. chabaudi chabaudi (AS or CB lines) or Plasmodium berghei (KSP-11 line) by repeated infection with homologous parasites. Serum from P. chabaudi chabaudi AS hyperimmune mice reacted with antigens released from disrupted P. chabaudi chabaudi AS-infected erythrocytes, but P. chabaudi chabaudi CB and P. berghei KSP-11 hyperimmune serum also contained cross-reactive antibodies to these antigens. However, antibody activity directed against antigens exposed at the surfaces of intact P. chabaudi chabaudi-infected erythrocytes was mainly parasite species specific and, to a lesser extent, parasite line specific. Importantly, this response included opsonizing antibodies, which bound to infected erythrocytes, leading to their phagocytosis and destruction by macrophages. The results are discussed in the context of the role that antibodies to both variable and invariant antigens may play in protective immunity in the face of continuous susceptibility to reinfection.     

von Willebrand Factor A domain-related protein, a novel microneme protein of the malaria ookinete highly conserved throughout Plasmodium parasites.

The mosquito-invasive form of the malarial parasite, the ookinete, develops numerous secretory organelles, called micronemes, in the apical cytoplasm. Micronemal proteins are thought to be secreted during midgut invasion and to play a crucial role in attachment and motility of the ookinete. We found a novel ookinete micronemal protein of rodent malarial parasite Plasmodium berghei, named P. berghei von Willebrand factor A domain-related protein (PbWARP), and report it here as a putative soluble adhesive protein of the ookinete. The PbWARP gene contained a single open reading frame encoding a putative secretory protein of 303 amino acids, with a von Willebrand factor type A module-like domain as a main component. Western blot analysis demonstrated that PbWARP was firstly produced 12 h after fertilization by maturing ookinetes as SDS-resistant complexes. Recombinant PbWARP produced with a baculovirus system also formed SDS-resistant high-order oligomers. Immuno-electron microscopic studies showed that PbWARP was randomly distributed in the micronemes. PbWARP homologues also exist in human malarial parasites, Plasmodium falciparum and Plasmodium vivax. Highly conserved primary structures of PbWARP homologues among these phylogenetically distant Plasmodium species suggest their functional significance and the presence of a common invasion mechanism widely utilized throughout Plasmodium parasites.     

Susceptibility of CXB recombinant inbred mice to murine plasmodia

The genetic control of susceptibility to two species of murine malarial parasites was investigated with recombinant inbred (RI) mouse strains as a model system. Initially, the nonlethal Plasmodium yoelii 17X strain was cloned by limiting dilution to minimize parasite variability. This cloned P. yoelii was used to infect C57BL and BALB/c mice, strains which are the progenitors of the CXB RI strains. Since these two strains displayed consistent differences in the kinetics of parasitemia, the seven CXB RI strains were compared for their susceptibility to the same parasite. The RI strains varied considerably when infected with P. yoelii and could be divided into susceptible and resistant groups based on mortality observed with this normally mild infection. This suggests a complex, multigenic inheritance determining susceptibility to this parasite. However, when the susceptible and resistant CXB RI mice were infected with another, unrelated plasmodial species, Plasmodium chabaudi adami, all the mice showed identical patterns of disease. Since susceptibility to different murine plasmodia does not cosegregate in the CXB RI mice, different mechanisms of resistance may be required for different plasmodial species.     

Primary sequences of two small subunit ribosomal RNA genes from Plasmodium falciparum

We have determined the complete sequence of two structurally distinct 18S ribosomal RNA genes from the malarial parasite Plasmodium falciparum. S1 nuclease analyses demonstrate that only one of the genes is represented in stable rRNA populations isolated from blood-stage parasites. Comparisons of homologous rRNA genes from Plasmodium berghei and P. falciparum reveal that they are identical at 86% of their positions. From comparisons of the Plasmodium genes to that of humans, it was possible to design genus-specific as well as species-specific oligonucleotide probes that can be used to distinguish the parasite 18S ribosomal RNA from that of its host. The utilization of these probes as diagnostic reagents is discussed.     

Impairment of Plasmodium falciparum-specific antibody response in severe malaria.

Serum antibody response to plasmodial antigens was investigated in 97 Thai patients with Plasmodium falciparum malaria. No difference in immunoglobulin G (IgG) antibody levels was detected between groups without or with cerebral manifestations of malaria (n = 40). In patients with the most severe form of the disease, i.e., those who died despite adequate therapy (n = 12), antibody detected in the immunofluorescent-antibody test was found at lower levels than in those who recovered (geometric means: IgG = 1/420 versus 1/3,800; IgM = 1/15 versus 1/70); similarly, precipitating malarial antibodies were present in only 1 of these 12 patients, while they were detectable in 65 of the remaining 85 patients (76.5%). In contrast, anticytomegalovirus antibody levels were similar in the different groups of patients. Results show that depression of antibody response may extend to antiplasmodial responses during severe malaria. The link between fatality and a low level of antibody production suggests that an appropriate immune response to malarial antigens may be required to achieve recovery with drug treatment and provides a new direction for malaria therapy research.     

A recombinant 15-kilodalton carboxyl-terminal fragment of Plasmodium yoelii yoelii 17XL merozoite surface protein 1 induces a protective immune response in mice.

Since the developmental stages of malarial parasites which replicate within erythrocytes are responsible for the morbidity and mortality associated with this disease, antigens produced by these stages have been proposed as candidates for a vaccine. One surface protein of merozoites (MSP-1) has been shown to immunize both rodents and primates against virulent challenge infection in experimental systems. However, little is known of relevant epitopes on the molecule, and attempts to obtain recombinant MSP-1 polypeptides in a native configuration have proven difficult. We have found that the cysteine-rich, carboxyl-terminal region of the MSP-1 protein from the rodent malarial parasite Plasmodium yoelii yoelii can be expressed in a native configuration as a fusion protein in Escherichia coli. This recombinant polypeptide containing 15 kDa of the predicted 197-kDa protein elicits antibodies in mice which recognize the native parasite MSP-1. Most significantly, both inbred and outbred mice immunized with the fusion protein in Ribi adjuvant are partially and in some cases completely protected against challenge infection with an otherwise lethal parasite strain. This is the first observation of such significant protection obtained with a small portion of the MSP-1 produced in recombinant systems.     

Accessibility and distribution of intraerythrocytic antigens ofPlasmodium-infected erythrocytes following mild glutaraldehyde fixation and detergent extraction

Malarial antigens on the surface of infected erythrocytes have been described by many investigators. However, few of these antigens have been unambiguously demonstrated to be exposed on the surface of erythrocytes. This study demonstrates that mild glutaraldehyde fixation results in the cytoplasmic face of the host membrane becoming accessible to antibody under conditions that normally do not expose the cytoplasmic face of uninfected erythrocytes. These results indicate that caution should be used in interpreting data on the membrane disposition of malarial antigens. Detergent extraction of the glutaraldehyde-fixed erythrocytes results in an increased permeabilization such that malarial antigens on the parasite surface and within the cytoplasm of the infected erythrocyte are accessible to antibody. The accessibility of these antigens was demonstrated by both immunofluorescence and two-color flow cytometry. The antigens within the host cytoplasm were not diffuse but associated with patchy aggregates. Analysis of the antigens associated with the cytoplasmic aggregates by immunoelectron microscopy indicated that they were not associated with membrane-bound compartments. The fixation and permeabilization protocol described herein will have useful applications for the characterization and analysis of malarial antigens.Abbreviations Ag antigen - FITC fluorescein isothiocyanate - HBSS Hanks' balanced salt solution - mAb monoclonal antibody - MSP1 merozoite surface protein 1 - PBS phosphate-buffered saline - PfHRPII Plasmodium falciparum histidine-rich protein II - RESA ring-stage erythrocyte surface antigen     

Identification of cross-reactive promastigote cell surface antigens of some leishmanial stocks by 125I labeling and immunoprecipitation.

Externally oriented surface membrane constituents of promastigotes from several Leishmania species were radiolabeled with 125I. Autoradiographs of cell surface-labeled and sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated proteins of the stocks revealed distinctive patterns of bands in the molecular weight range of 6,000 to 240,000. Immunoprecipitation of detergent extracts of the labeled promastigote stocks with anti-Leishmania donovani membrane serum demonstrated that each of the stocks contained some antigenically cross-reactive determinants. The electrophoretic patterns of these determinants serve both to distinguish the parasite stocks (by unique, species-specific patterns) and to indicate antigenic similarities in stocks thought to be different by other biochemical criteria. At least 12 cross-reactive cell surface antigens in two New World leishmanias are recognized by polyvalent anti-L. donovani serum, suggesting that these common leishmanial antigens may account for the documented serological cross-reactivities among various Leishmania species. In all stocks tested, an iodinated protein was identified which had a relative molecular weight of 65,000 under reducing conditions but which demonstrated an increase in relative mobility in sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions. Distinctive patterns of the antigens common to the several stocks were also demonstrated with the use of monoclonal antibodies.     

Truncation of Plasmodium berghei merozoite surface protein 8 does not affect in vivo blood-stage development

Merozoite surface protein 8 (MSP8) has shown promise as a vaccine candidate in the Plasmodium yoelii rodent malaria model and has a proposed role in merozoite invasion of erythrocytes. However, the temporal expression and localisation of MSP8 are unusual for a merozoite antigen. Moreover, in Plasmodium falciparum the MSP8 gene could be disrupted with no apparent effect on invitro growth. To address the invivo function of full-length MSP8, we truncated MSP8 in the rodent parasite Plasmodium berghei. PbDeltaMSP8 disruptant parasites displayed a normal blood-stage growth rate but no increase in reticulocyte preference, a phenomenon observed in P. yoelii MSP8 vaccinated mice. Expression levels of erythrocyte surface antigens were similar in P. berghei wild-type and PbDeltaMSP8-infected erythrocytes, suggesting that a parasitophorous vacuole function for MSP8 does not involve global trafficking of such antigens. These data demonstrate that a full-length membrane-associated form of PbMSP8 is not essential for blood-stage growth.     

Sera of patients with systemic lupus erythematosus react with plasmodial antigens and can inhibit the in vitro growth of Plasmodium falciparum

The acquisition of protective immunity in malaria is a slow process during which autoantibodies are produced. The present work aimed at studying a possible interference of autoimmune responses on malaria immune protection. This was done by investigating the presence of autoantibodies in the sera of malarious patients, by searching for reactivity of autoantibodies from autoimmune patients against plasmodial antigens, and by studying the effect of such antibodies on the in vitro growth of Plasmodium falciparum. Sera from systemic lupus erythematosus (SLE) and malaria patients were tested against autologous and plasmodial antigens. Out of the 109 SLE sera tested, 48 (44%) reacted against the parasite. In addition, 26 (47%) out of 55 randomly selected sera, mainly those containing anti-DNA and antinuclear autoantibodies, were able to inhibit parasite growth to some extent. Conversely, a high frequency (81%) of sera of malaria patients exhibited reactivity against autoantigens. The results show that patients with autoimmune processes can produce antibodies that recognize plasmodial antigens in the absence of plasmodial infection, that malaria patients can produce autoantibodies, that SLE sera can inhibit plasmodial growth in vitro, and that the presence of anti-DNA and antinuclear antibodies may be important in such anti-plasmodial activity. It is concluded that autoimmune responses may have influence on the protective immunity against malaria.     

Plasmodium falciparum: characterization of defined antigens by monoclonal antibodies.

Monoclonal antibodies directed against Plasmodium falciparum detect stage-specific, species-specific and common antigenic determinants of Plasmodia. These antibodies provide new tools for purification and characterization of Plasmodium falciparum antigens in relation to future procedures for immunoprophylaxis.     

The movement of fluorescent endocytic tracers in Plasmodium falciparum infected erythrocytes.

The fluorescent lipophilic probe 1,1'-dihexadecyl-3-3'-3-3'- tetramethylindocarbocyanine (diIC16) inserted in the red cell surface, functioned as a non-exchangeable lipid marker which was not metabolised or toxic in plasmodial cultures. Invasion by Plasmodium falciparum resulted in the internalisation of the lipid, suggesting the uptake of red cell membrane components during parasite entry. The fluorescent lipid was not transported from red cell to parasite membranes at subsequent stages of development, but label in the erythrocyte-derived parasitophorous vacuole was eventually detected in daughter merozoites. Fluorescent dextrans of 10 kDa in the extracellular medium were also not internalised during intraerythrocytic parasite growth. The results support that with the exception of the invasion step, plasmodial infection does not induce endocytosis in the erythrocyte membrane. Despite the lack of endocytosis, both D and L stereoisomers of the head group blocked phospholipid analogue N-4-nitrobenzo-2-oxa-1,3-diazoledipalmitoyl phosphatidylethanolamine (N-NBD-PE) inserted in the erythrocyte membrane, were internalised by mature infected erythrocytes. Lipid internalisation occurred by a non head group dependent parasite mechanism, which could account for the stage-specific uptake of phospholipids observed in mature infected erythrocytes. We were unable to detect the transport of carbocyanine dyes and N-NBD-PE from intracellular parasites back to the erythrocyte membrane. Additionally, the carbocyanine dyes were not transferred between adjacent organisms in a double infected red cell. The data argue for the absence of bulk membrane lipid transport between individual parasites and their host cell bilayer in an infected erythrocyte.     

Persistent histidine-rich protein 2, parasite lactate dehydrogenase, and panmalarial antigen reactivity after clearance of Plasmodium falciparum monoinfection

We tested 240 patients with Plasmodium falciparum monoinfection for persistent parasite antigenemia after successful standardized antimalarial therapy by using the ICT Malaria Pf/Pv and OptiMAL-IT assays that detect the malaria antigens Plasmodium falciparum histidine-rich protein 2 (HRP2) and parasite lactate dehydrogenase (pLDH), respectively, as well as a panmalarial antigen (PMA). The patients were screened for antigenemia on days 0, 3, 7, and 14 of follow-up. On day 0, all 240 patients showed positive reactivity with both assays. Of the 229 cases with negative parasitemia on day 3, persistent antigenemia was observed in 207 (90.4%) of the cases: 188 (82.1%) for HRP2 antigen and 75 (32.8%) for PMA. There was a gradual decrease in antigenemia on follow-up to day 14; however, the drop in reactivity to PMA was less than that for HRP2 antigen. In contrast to HRP2 antigenemia, there was a significant decrease in pLDH antigenemia to 38.4% and to 14.8% (PMA) on day 3 (P < 0.03). The pLDH antigenemia level dropped further to 14.8% on day 7. There was no significant association of persistent antigenemia with gametocytemia. One case with gametocytemia was negative for both the antigens. In conclusion, the OptiMAL-IT assay is more sensitive than the ICT Malaria Pf/Pv test for monitoring therapeutic responses after antimalarial therapy since the LDH activity ceases when the malarial parasite dies.     

The characterization of two monoclonal antibodies which react with high molecular weight antigens of asexual Plasmodium falciparum.

We prepared rat monoclonal antibodies (mAb) specific for very large Plasmodium falciparum proteins to assist in their characterization. Hybridomas prepared from rats immunized with parasitized erythrocyte (PE) proteins of greater than 200 kDa exhibited two patterns of Western blot reactivity with PE SDS extracts: one represented by clone 41E11 (IgM, kappa), the other by clone 12C11 (IgM, lambda). MAb 41E11 reacted by Western blotting with at least 15 antigens, most of which comigrated with antigens identified by the 33G2 human IgM mAb. The stage specificity of mAb 41E11 reactivity and indirect immunofluorescence (IFA) pattern closely resemble those previously described for antigens that share the EEXXEE sequence motif. Unlike mAb 33G2, MAb 41E11 immunoprecipitated a biosynthetically radiolabeled protein of 320 kDa. MAb 41E11 did not immunoprecipitate any cell surface 125I proteins. MAb 12C11 reacted on Western blotting with a different group of malarial antigens of approximately 44, 95, 117, 145, and 310 kDa, as well as with some low-molecular-weight, uninfected erythrocyte antigens. MAb 12C11 did not immunoprecipitate any cell surface 125I or biosynthetically labeled proteins. The 310-kDa antigen recognized by mAb 12C11 (denoted Ag 12A) does not correspond to PfEMP2 or the 320-kDa antigen recognized by mAbs 33G2 or 41E11. With trophozoites and more mature stages, fixed IFA reactivity of mAb 12C11 was at the parasite and in antigen aggregates in the host cell cytoplasm that extended to the PE plasma membrane. Indirect results suggest that Ag 12A does not correspond to cell surface-exposed PfEMP1 and is most likely a hitherto unidentified malarial protein.     

Identification of a Plasmodium chabaudi antigen present in the membrane of ring stage infected erythrocytes

A novel antigen of asexual blood stages of the rodent malaria parasite Plasmodium chabaudi, was detected by means of a modified indirect immunofluorescence assay (IFA), using glutaraldehyde fixed and air dried monolayers of P. chabaudi infected erythrocytes. P. chabaudi hyperimmune sera gave a distinct surface immunofluorescence of erythrocytes infected with early stages of the parasite. Fixation and drying of the erythrocytes was necessary for the antigenic activity to be exposed. The antigens were species specific as P. chabaudi hyperimmune serum only stained P. chabaudi but not P. yoelii or P. falciparum infected erythrocytes. The antigenic activity involved in the IFA was resistant to trypsin, phospholipases and neuraminidase but not to pronase, suggesting that the antigens were polypeptides. The surface immunofluorescence was inhibited by an extract of parasitized erythrocytes, but not by similar extracts of normal erythrocytes. The inhibitory antigens were soluble and heat stable (100 degrees C, 5 min). For identification and characterization of the antigens, antibodies were isolated by acid elution from monolayers of infected erythrocytes and monoclonal antibodies were produced. Probing in immunoblotting of extracts of parasitized erythrocytes separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis with the eluted antibodies, showed that they reacted consistently with a polypeptide of Mr 105 000 (Pch105). The Pch105 antigen shares many characteristics with Pf155, a P. falciparum antigen considered as a candidate for a vaccine against that parasite.     

Antigenic relationships between the surface-exposed, secreted and somatic materials of the nematode parasites Ascaris lumbricoides, Ascaris suum, and Toxocara canis.

The cosmopolitan nematode parasites Ascaris lumbricoides, Ascaris suum, and Toxocara canis are closely related phylogenetically, and are all pathogenic to man. In the case of the latter, the antigens released by the tissue-invasive parasitic larvae in vitro ('excretory/secretory' or 'ES' antigens) are routinely used for serodiagnostic purposes. Here we have found, using radioimmunoprecipitation with defined rabbit antiserum, and SDS-PAGE, that there is a significant antigenic similarity between the secreted and somatic antigens of the three nematodes, and have characterized cross-reactive components. Among these is a 14 kD internal protein which has a homologue in all three parasites. This molecule is the subject of an IgG antibody response in Ascaris infection, but there is no measurable response to it in toxocariasis. Lastly, using quantitative immunofluorescence, the antigens exposed on the surface of intact, living, larvae were found to be cross-reactive or specific depending on the developmental stage of the parasites. This means that the surface of tissue-invasive Ascaris larvae bears stage-specific epitopes.     

Immunity to blood-stage murine malarial parasites is MHC class II dependent

To determine whether MHC class II antigen presentation is essential for the induction of protective immunity against blood-stage malarial parasites, we used gene-targeted knockout (KO) mice to follow the time-course of nonlethal Plasmodium yoelii and Plasmodium chabaudi infections in two models of MHC class II deficiency. Infection of MHC class II KO (A(-/-)) mice with either parasite species resulted in an unremitting hyperparasitemia, whereas MHC-intact control mice resolved their parasitemia. In contrast, invariant chain KO (Ii(-/-)) mice, which present antigen via recycled but not nascent MHC class II molecules, eventually cured their infections when infected with P. yoelii. P. chabaudi parasitemia declined to subpatent levels in most Ii(-/-) mice but then recrudesced. Immunity to blood-stage malaria may be achieved by cell-mediated and antibody-mediated mechanisms of immunity, as such, the findings in A(-/-) mice indicate an essential role for MHC class II presentation of malarial antigens. Moreover, they suggest that protective immune responses to malarial antigens capable of eliminating blood-stage parasites are T cell dependent and can be induced with antigens processed in early and late endosomes.     

Isolation and analysis of nephritic-producing immune complexes in Plasmodium berghei-infected mice.

A nephritic condition was developed by infecting Swiss Webster albino mice with the malarial parasite Plasmodium berghei NK 65. These animals were tested for urinary protein and the presence of circulating immune complexes using reagent strips and a polyethylene glycol (PEG) precipitation assay. The circulating immune complexes were isolated from the sera using both affinity chromatography and PEG precipitation and from the kidney by acid elution. The isolated complexes were dissociated into their individual components and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The components of the complexes were transferred to nitrocellulose sheets and probed for the presence of malarial antigens using a rabbit anti-P berghei antisera. The overall humoral response to the malarial parasite was evaluated using a radial immunodiffusion assay. The present study confirmed that the malarial-infected animals not only developed the nephritic condition (as evident by the high levels of proteinuria) but also, as indicated by the PEG assay, have the presence of high levels of circulating immune complexes in their serum. The apparent absence in the SDS gels of any abnormal protein bands followed by the inability of the Western blot to reveal any malarial antigens provides some of the strongest evidence to date that these malarial proteins are not directly involved in the circulating immune complexes believed to be responsible for producing this nephritic condition.     

High-Resolution Autoradiography of Malarial Parasites Treated with 3H-Chloroquine

Electron microscope autoradiography was performed on the erythrocytic stages of the rodent malarial parasite, Plasmodium berghei, after exposure to ³H-Chloroquine. ³H-Chloroquine becomes selectively localized within the parasite food vacuoles 1 hour after it is administered, and remains in this structure for up to 24 hours. This study indicated that the food vacuoles were the primary site of chloroquine concentration within the parasite.